Association of specific mutations in katG, rpoB, rpsL and rrs genes with spoligotypes of multidrug-resistant Mycobacterium tuberculosis isolates in Russia.

Most multidrug-resistant (MDR) Mycobacterium tuberculosis isolates in Russia belong to the Beijing or Latino-American and Mediterranean (LAM) spoligotype families. The objective of this study was to investigate possible associations between genotype and the frequencies of mutations that confer drug resistance in a population that has two large families of circulating strains. Spoligotyping, IS6110 restriction fragment length polymorphism typing, and sequencing of the katG and rpoB genes, were performed for 217 consecutive MDR M. tuberculosis isolates from patients. The rpsL and rrs genes were also sequenced for selected streptomycin-resistant isolates. Of the 217 MDR isolates, 99 (46%) belonged to the LAM family, 92 (42%) to the Beijing family, 21 (10%) to the Haarlem family and four (2%) to the T family. There was one unique spoligotype. Mutations in the katG gene were identified in 207 (95%) isolates, all of which had mutations in codon 315. Mutations in the rpoB gene were identified in 200 (92%) isolates; 75% of LAM isolates carried a mutation in codon 516, whereas 71% of Beijing isolates carried a mutation in codon 531. In the 33 isolates resistant to streptomycin 50 mg/L, the 43AGG rpsL mutation was found in 27% of Haarlem, 75% of Beijing and 0% of LAM isolates, and rrs mutations were found in 17% (516C-->T) of Beijing and 100% (513A-->C) of LAM isolates. Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampicin or streptomycin in the Beijing and LAM families. The biological implications of this correlation remain to be explored.

Characterization of drug-resistant isolates of Mycobacterium tuberculosis derived from Russian inmates.

SETTING: Tuberculosis ward of a prison in Russia. OBJECTIVE: Molecular characterization of drug-resistant isolates. DESIGN: Isolates were collected from all tuberculosis patients occurring in the prison over a 1-year period. RESULTS: Of 130 patients studied, 17 patients produced pan-susceptible isolates and 113 produced isolates resistant to at least one drug, including 85 multidrug-resistant isolates. Mutations at katG315 occurred in 98% of isoniazid-resistant isolates. Mutations in rpoB were found in 89% of rifampicin-resistant isolates. Mutations in pncA occurred in 13% of the 75 isolates tested. By spoligotyping, members of the Beijing (55 isolates) and LAM (31 isolates) families were identified. By IS6110 genotyping, two groups (34 and 55 isolates) of related isolates were found, including three clusters (10, 12, and 16 isolates) with identical patterns. In a study of samples collected 3 months apart from 28 patients, four patients produced isolates containing a mixture of strains and five patients produced specimens containing distinctly different isolates. Isolates of nine patients acquired additional drug resistance. CONCLUSION: Three families of strains accounted for much of the drug-resistant tuberculosis in this population. Multiple resistance, acquisition of resistance, and infection with two or more strains as well as reinfection were observed.

Identification of two Mycobacterium tuberculosis H37Rv ORFs involved in resistance to killing by human macrophages.

BACKGROUND: The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times. RESULTS: Two of the clones recovered from this enrichment process, sur2 and sur3, exhibited significantly increased survival relative to wild-type bacteria. In coinfection experiments, the ratio of sur2 colonies to wild-type colonies was 1:1 at 0 hours but increased to 20:1 at 24 hours post phagocytosis. The ratio of sur3 colonies to wild-type colonies was 1:1 at 0 hours and 5:1 at 24 hours. The M. tuberculosis ORFs responsible for increased survival were shown to be Rv0365c for the sur2 clone and Rv2235 for the sur3 clone. These ORFs encode proteins with as-of-yet unknown functions. CONCLUSIONS: We identified two M. tuberculosis ORFs which may be involved in the ability of tubercle bacilli to survive in macrophages.

Diagnostic test needs for evaluating antituberculosis vaccines.

To aid in the evaluation of preexposure and postinfection vaccines to prevent tuberculosis, diagnostic tests are needed that can clearly distinguish immunologic protection from vaccine failure in a timely manner. The currently available tests to detect infected persons (tuberculin skin-test) and confirm active disease (conventional culture methods) have limitations in specificity, sensitivity, or timeliness. Research to identify (1) surrogate markers of infection, disease, or protection and (2) stage-specific antigens or immune responses holds some promise for the development of new tests that can distinguish the various outcomes of an infection or a vaccination.

Evaluation of Mycobacterium tuberculosis genes involved in resistance to killing by human macrophages.

A coinfection assay was developed to examine Mycobacterium tuberculosis genes suspected to be involved in resistance to killing by human macrophages. THP-1 macrophages were infected with a mixture of equal numbers of recombinant Mycobacterium smegmatis LR222 bacteria expressing an M. tuberculosis gene and wild-type M. smegmatis LR222 bacteria expressing the xylE gene. At various times after infection, the infected macrophages were lysed and the bacteria were plated. The resulting colonies were sprayed with catechol to determine the number of recombinant colonies and the number of xylE-expressing colonies. M. smegmatis bacteria expressing the M. tuberculosis glutamine synthetase A (glnA) gene or open reading frame Rv2962c or Rv2958c demonstrated significantly increased survival rates in THP-1 macrophages relative to those of xylE-expressing bacteria. M. smegmatis bacteria expressing M. tuberculosis genes for phospholipase C (plcA and plcB) or for high temperature requirement A (htrA) did not.

Identification of promiscuous epitopes from the Mycobacterial 65-kilodalton heat shock protein recognized by human CD4(+) T cells of the Mycobacterium leprae memory repertoire.

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4(+) T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosis HSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae and M. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4(+) T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.

Multiplex PCR assay to aid in the identification of the highly transmissible Mycobacterium tuberculosis strain CDC1551.

SETTING: Mycobacterium tuberculosis strain CDC1551 outbreak area in Tennessee and Kentucky and selected locations in the USA. OBJECTIVE: Develop a PCR assay to distinguish the highly transmissible CDC1551 from strains which have similar 4-band IS6110 fingerprints. DESIGN: Compare the IS6110 insertion sites in CDC1551 with those in 10 isolates which have similar 4-band IS6110 fingerprints. Utilize unique characteristics of insertion sites in CDC1551 to design a multiplex PCR to identify this strain. RESULTS: A multiplex PCR was developed which targets an IS6110 insertion conserved in most IS6110 low copy number strains and a deletion within the direct repeat region adjacent to an IS6110 insertion. Of 139 isolates with similar 4-band fingerprints, the CDC1551 PCR pattern was generated by only the 14 outbreak associated isolates. Of 154 isolates with different fingerprints, only four generated the CDC1551 pattern and these could be distinguished from CDC1551 by their IS6110 fingerprint. CONCLUSIONS: The multiplex PCR used in conjunction with the IS6110 fingerprint should be a useful tool to aid in the continued surveillance of the outbreak area and follow the spread of this highly transmissible strain of M. tuberculosis.

IS1549 from Mycobacterium smegmatis forms long direct repeats upon insertion.

A new insertion element, IS1549, was identified serendipitously from Mycobacterium smegmatis LR222 during experiments using a vector designed to detect the excision of IS6110 from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the aph gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS231, which is a member of the IS4 family of insertion elements. IS1549 contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS1549 are present in LR222, but not all M. smegmatis strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of M. smegmatis 607 contain IS1549. None of 13 other species of Mycobacterium tested by PCR with two sets of primers specific for IS1549 were positive for the expected amplified product.

The mtp40 gene is not present in all strains of Mycobacterium tuberculosis.

A multiple PCR-based assay that targets IS6110 and the mtp40 gene was evaluated for the rapid differentiation of Mycobacterium bovis and M. tuberculosis, two of the causative agents of tuberculosis. The IS6110 target is present in both species, whereas the mtp40 gene was thought to be specific for M.tuberculosis (P. Del Portillo, L.A. Murillo, and M.E. Patarroyo, J. Clin. Microbiol. 29:2163-2168, 1991). However, the mtp-40 gene is not present in all M. tuberculosis strains and, hence, is not useful for differentiating M.tuberculosis and M.bovis.

HLA-DR4-restricted T-cell epitopes from the mycobacterial 60,000 MW heat shock protein (hsp 60) do not map to the sequence homology regions with the human hsp 60.

The mycobacterial 60,000 MW heat shock protein (hsp 60) is a major antigen recognized by mycobacteria-reactive human CD4+ T cells with lymphokine profiles and effector functions consistent with protective immunity. In addition, the presence of a large number of T-cell epitopes presented by several HLA class II molecules makes this antigen relevant to subunit vaccine design. However, the results from animal models as well as human studies suggest that the mycobacterial hsp 60 may induce T-cell-mediated autoimmune conditions. In humans, the expression of HLA-DR4 represents a risk factor for some autoimmune diseases. These observations suggest that the epitopes from the mycobacterial hsp 60 presented to T cells in the context of HLA-DR4 could be relevant to autoimmunity. This is the first report on identification of HLA-DR4-restricted T-cell epitopes from the mycobacterial antigen hsp 60. In total, five epitopes recognized in the context of HLA-DR4 by the M. leprae hsp 60-reactive CD4+ T-cell clones from a subject immunized with M. leprae were defined by synthetic peptides. Two of the epitopes were M. leprae-specific (aa 343-355, aa 522-534), whereas three epitopes were common to M. leprae and M. tuberculosis (aa 331-345, aa 441-455, aa 501-515). However, all of these epitopes belong to the regions that are highly divergent between the mycobacterial hsp 60 and the homologous human hsp 60 sequence, suggesting that the T cells recognizing the mycobacterial hsp 60 in the context of HLA-DR4 may not necessarily induce autoreactivity.

Anti-Hsp65 antibodies recognize M proteins of group A streptococci.

Group A streptococcal M protein and the mycobacterial heat shock protein, hsp65, are strong bacterial immunogens that have been linked to arthritis and autoimmunity. Recent evidence has shown that streptococcal arthritis and adjuvant arthritis may be related to epitopes shared between group A streptococci and hsp65. We investigated the possibility that immunological similarities were shared between streptococcal M protein and hsp65. Antibodies against the 65-kDa heat shock protein of Mycobacterium tuberculosis were tested for reactivity with group A streptococci and purified recombinant M proteins (rM5 and rM6). Rabbit polyclonal anti-hsp65 serum was highly reactive with M type 5 Streptococcus pyogenes and rM5 and rM6 proteins in an enzyme-linked immunosorbent assay (ELISA). A mouse anti-hsp65 monoclonal antibody (MAb), IIC8, reacted with streptococcal M types 5, 6, 19, 24, and 49 in an ELISA but showed no reactivity with an isogenic streptococcal mutant which did not express M protein. Anti-hsp65 MAb IIC8 recognized rM5 and rM6 proteins in the ELISA, and MAbs IIC8 and IIH9 reacted strongly with rM6 protein in Western immunoblots. The binding of M protein by anti-hsp65 MAbs was shown to be inhibited by both hsp65 and M protein. These data show that anti-hsp65 antibodies recognize streptococcal M proteins.

Regulation of murine macrophage IL-12 production. Activation of macrophages in vivo, restimulation in vitro, and modulation by other cytokines.

IL-12 is important in the host response to a variety of pathogens. It plays an adjuvant-like role in an initial immune response as well as a therapeutic role in established infections. Despite its well documented importance, comparatively little is known about the regulation of IL-12 production. In this study, we examined IL-12 production by cultured murine peritoneal macrophages from two perspectives: 1) macrophage activation in vivo, and 2) stimulation of IL-12 secretion in vitro. Macrophages were maximally activated within 48 h in vivo during infection with Listeria. Interestingly, although avirulent or heat-killed Listeria induced only minimal production of IL-12 by macrophages, the immunogenic combination of heat-killed bacteria and rIL-12 was highly stimulatory for IL-12 production. LPS and peritoneal inflammatory agents were also stimulatory, but latex beads were ineffective, indicating that microbial components were essential and phagocytosis alone was insufficient. Restimulation in vitro revealed similar patterns, in that infection and LPS were stimulatory but latex beads were not. A systematic survey of potential stimulatory agents showed that microbial heat shock proteins, crude bacterial extracts, bacterial superantigens, a yeast extract, and dsRNA induced IL-12 in vitro. Other cytokines also influenced IL-12 induction. IFN-gamma, which is up-regulated during infection, acted in synergy with other stimuli, suggesting an amplification loop for IL-12 production, whereas IL-4, IL-10, IL-13, and TGF-beta were inhibitory. The existence of a broad range of stimuli from a wide variety of pathogenic organisms underscores the fundamental importance of IL-12 in host defense.

Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy.

During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.

Diagnostic mycobacteriology laboratory practices.

The resurgence of tuberculosis has forced clinical laboratories to improve the methods used for detection of M. tuberculosis. Current recommendations for diagnostic laboratory performance [7] include (1) daily processing of specimens (i.e., handling these specimens in the same way that all other specimens sent to the laboratory are handled); (2) inoculation of liquid media (e.g., BACTEC) for the primary culture; (3) use of nucleic acid probes or the NAP test for identifying isolates as M. tuberculosis as soon as possible; (4) determining drug susceptibility with use of liquid media; and (5) reporting results of each step to physicians in a timely manner. The immediate goals are to report identification of M. tuberculosis within 10-14 days of receipt of the specimen and to report drug susceptibilities within 15-30 days. This can be done if current technologies are fully utilized. The amplification-based systems for the identification of M. tuberculosis and the luciferase-based systems for rapid determination of drug susceptibilities should help further shorten turn-around times. The results to date demonstrate that these systems are feasible, although they must be reduced to formats that can be used routinely in clinical laboratories. The gene-amplification systems may be the most promising, and they are nearing commercial availability. If the assays function as well during routine use as they have during clinical trials, a clinical laboratory may soon be able to report confirmed M. tuberculosis infection to the physician within hours of receiving a specimen, instead of within the typical period of 2-4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular biology, virulence, and pathogenicity of mycobacteria.

The diseases resulting from infections with Mycobacterium species are important sources of morbidity and mortality throughout the world today, with particularly devastating effects in tropical and developing countries. Almost 2 billion people have been infected with Mycobacterium tuberculosis, the causative agent of tuberculosis, and approximately 3 million people die each year from this disease. Tuberculosis also has re-emerged as an important public health problem in the United States, and this resurgence has been accompanied by an increased incidence of tuberculosis resistant to the standardly used anti-tuberculosis drugs. Researchers' ability to investigate the molecular basis of the pathogenicity and drug resistance of the mycobacteria has been hampered by a lack of appropriate experimental tools. However, during the past 5 years, tremendous progress has been made in the development of the molecular biology of mycobacteria, and molecular tools are now available for detailed analysis of their genetics and for elucidation of the molecular mechanisms of their pathogenicity. The development of these tools is briefly reviewed, and the uses of the tools to investigate drug resistance in Mycobacterium tuberculosis, to identify mycobacterial virulence factors, and to explore intracellular survival strategies are described.

Rapid Mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing.

OBJECTIVE: To develop and demonstrate the utility of automated DNA sequencing strategies for rapid and unambiguous identification of Mycobacterium species and mutations associated with antimicrobial resistance in Mycobacterium tuberculosis. DESIGN AND SPECIMENS: A 360-base pair segment of the gene (hsp65) encoding a 65-kd heat shock protein was characterized from 91 isolates assigned to 24 Mycobacterium species by traditional biochemical techniques. Areas of seven genes recently shown to contain mutations associated with antimicrobial resistance in M tuberculosis strains were also sequenced in a sample of 128 resistant organisms. Early positive BACTEC 460 cultures and acid-fast, bacterium-positive sputum specimens from patients with tuberculosis were also studied. RESULTS: Automated DNA sequencing identified species-specific polymorphism in the target segment of hsp65, successfully identified organisms to the species level in smear-positive sputum samples, and unambiguously characterized seven genes associated with antimicrobial resistance in M tuberculosis. CONCLUSIONS: Rapid identification of M tuberculosis and other Mycobacterium species is possible by automated DNA sequencing of a portion of hsp65. The technique is also feasible for analysis of some smear-positive sputum specimens. Unambiguous characterization of target segments of genes harboring mutations associated with antimicrobial resistance in M tuberculosis is possible from primary patient specimens. Taken together, the data demonstrate the feasibility of mycobacterial species identification and potential to identify mutations associated with antimicrobial resistance in less than 48 hours.